Expression, purification and crystallization of the glutathione-S-transferase E6(AsGSTE6) from <em>Anopheles sinensis</em> (Diptera: Culicidae)
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ACTA ENTOMOLOGICA SINICA  2018, Vol. 61 Issue (1): 59-67    DOI: 10.16380/j.kcxb.2018.01.007
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Expression, purification and crystallization of the glutathione-S-transferase E6(AsGSTE6) from Anopheles sinensis (Diptera: Culicidae)
MIAO Ya, ZHANG Tian-Tian, XU Bo-Ying*, CHEN Bin*
 (Chongqing Key Laboratory of Vector Insects, Institute of Insect and Molecular Biology, Chongqing Normal University, Chongqing 401331, China)
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Abstract 【Aim】 This study aims to explore the structure of the glutathione-S-transferase E6 (AsGSTE6) from Anopheles sinensis based on its preliminary crystallography. 【Methods】 AsGSTE6 was predicted and analyzed by bioinformatics method. AsGSTe6 was cloned, and the recombinant plasmid pET28a-AsGSTe6 was constructed and expressed in prokaryotic expression system. The recombinant protein was purified with nickel chelate affinity chromatography and gel filtration chromatography. The polymerization state of AsGSTE6 was detected by gel filtration chromatography and chemical crosslinking analysis. Crystal screening was performed by sitting drop vapor diffusion techniques. 【Results】 Bioinformatic analysis revealed that AsGSTE6 is a hydrophilic protein with the calculated molecular weight of 25.28 kD, without transmembrane regions and signal peptides in the amino acid sequence. The 3D structural prediction showed that AsGSTE6 contains a small N-terminal domain following a βαβαββα pattern and a large C-terminal all-α domain. The result of multiple sequence alignment demonstrated that GSTE6 proteins are highly conserved in insects. The 672 bp coding sequence of AsGSTe6 was cloned, and the recombinant plasmid pET28a-AsGSTe6 was constructed correctly. The recombinant protein AsGSTE6 was expressed in soluble form in Escherichia coli. And then the highly purified and stable protein was obtained with two step affinity chromatography. Gel filtration chromatography and chemical crosslinking analysis showed that AsGSTE6 mainly existed as dimer in vitro. Finally, the protein crystals of AsGSTE6 were obtained by crystal screening. 【Conclusion】 Crystals of AsGSTE6 have been obtained by methods of crystallography, which lays the foundation for 3D structure determination of AsGSTE6.
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MIAO Ya
ZHANG Tian-Tian
XU Bai-Ying
CHEN Bin
Key wordsAnopheles sinensis   glutathione-S-transferases   AsGSTE6   prokaryotic expression   purification   crystallization     
Cite this article:   
MIAO Ya,ZHANG Tian-Tian,XU Bai-Ying et al. Expression, purification and crystallization of the glutathione-S-transferase E6(AsGSTE6) from Anopheles sinensis (Diptera: Culicidae)[J]. ACTA ENTOMOLOGICA SINICA, 2018, 61(1): 59-67.
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http://www.insect.org.cn/EN/10.16380/j.kcxb.2018.01.007      or     http://www.insect.org.cn/EN/Y2018/V61/I1/59
 
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